Production of streptococcal M protein

ABSTRACT

Methods and compositions are provided for the cloning and expression of Streptococcus pyogenes M protein genes, and, in particular, types 5, 6 and 24 gene in single-cell host organisms. The streptococcal M protein produced by the recombinant DNA techniques described herein may be formulated for use as immunogens in vaccines to protect against S. pyogenes infections. The gene for the M protein may further be employed as a molecular probe for the accurate identification of streptococci in infected body tissues and fluids.

This application is a continuation application of Ser. No. 08/061,812filed on May 13, 1993, now abandoned, which is a continuationapplication of Ser. No. 07/800,890 filed on Nov. 27, 1991, nowabandoned, which is a continuation application of Ser. No. 07/604,353filed on Oct. 26, 1990, now abandoned, which is a continuation of Ser.No. 07/456,189 filed on Dec. 15, 1989, now abandoned, which is acontinuation of Ser. No. 07/141,586 filed on Jan. 7, 1988, nowabandoned, which is a divisional of Ser. No. 06/621,716 filed on Jun.18, 1984, now U.S. Pat. No. 4,784,948 which is a continuation-in-part ofSer. No. 06/521,962, filed on Aug. 10, 1983, now abandoned.

The invention described herein was made in the course of work supportedin part by grants from the National Institutes of Health of theDepartment of Health and Human Services.

1. FIELD OF THE INVENTION

This invention relates to compositions and processes for use asdiagnostic probes and for the production of vaccines utilizing as animmunogen an antiphagocytic streptococcal protein, such as M6 protein ofGroup A streptococcus. The M protein is a fibrillar surface moleculewhich enables streptococcus to resist phagocytosis by macrophages andpolymorpnonuclear neutrophiles of the infected host organism.

The present invention utilizes recombinant DNA techniques to insert aDNA sequence coding for M protein, or a portion thereof, into a DNAplasmid, such as viral DNA, plasmid DNA or cosmid DNA, such that theplasmid is capable of replicating and directing expression of the Mprotein gene in a bacterial host or other single cell system. Theresulting recombinant DNA molecules are introduced into host cells toenable production of M protein, or a portion or molecular variantthereof. The protein produced is then isolated, purifies and modifiedfor use as an immunogen in a vaccine against streptococcal infection.

This invention further provides a method for the detection of Group A, Cand G streptococci. Such detection is achieved through the use of aspecific molecular probe based on the entire gene or a segment of thegene coding for the M protein of Group A streptococcus. A DNA probeuseful in a hybridization screening method is described herein for theexamination of clinical isolates in cases of suspected streptococcalinfection.

2. BACKGROUND OF THE INVENTION

It is well recognized that acute rheumatic fever and acuteglomerulonephritis are sequelae of Group A streptococcal infection. Indeveloping countries of the tropics and subtropics, rheumatic heartdisease is currently the most common form of cardiac damage. Prevalencerates of this disease as high as 22-23 per thousand have been reportedin school-age children in urban slums of some developing countries ofthe world. It is estimated that in India alone, as many as six millionchildren may be afflicted. Although the exact mechanisms of diseasecausation are not understood, it is clear that rheumatic fever, as wellas acute nephritis, follows infection with Streptococcus pyogenes (GroupA streptococcus).

The streptococcal M protein is the major virulence factor of thisbacterium by virtue of the fact that it imparts to the organismresistance to phagocytic attack. Antigenic variation is the primarymechanism by which the Group A streptococcus is able to evade the host'simmune response and thus cause disease in man. Resistance to Group Astreptococcal infection is dependent upon the presence of type-specificantibodies to the M protein, a fibrillar molecule found on the surfaceof the organism. In addition to a number of nontypable strains, aboutseventy distinct Group A streptococcal M types are currently recognized.Despite the fact that antibodies cross-reactive among certain M typesare common, only antibodies prepared against the homologous type arecapable of initiating phagocytosis of the organisms (i.e., are opsonicantibodies). Furthermore, not all homologous, or type-specificantibodies are opsonic.

The fact that specific antiserum can be prepared to Group A streptococcihas made it possible to detect streptococcal infection by subjectingclinical isolates, such as those obtainable by throat swab, toserological testing. The identification of group A streptococci in aninfection requires the isolation of the organism in pure culture,extraction of the group-specific carbohydrate, and reaction withgroup-specific antiserum. A clinical test for streptococcal infectionthat could be based upon a property common only to all pathogenicstrains would thus be highly desirable.

2.1. RECOMBINANT DNA TECHNOLOGY AND GENE EXPRESSION

Recombinant DNA technology involves the technique of DNA cloning wherebya specific DNA fragment is inserted into a genetic element called avector which is capable of replication and transcription in the hostcell. The vector can be either a plasmid or a virus. Plasmids are small,circular molecules of double-stranded DNA that occur naturally in bothbacteria and yeast, where they replicate as independent units as thehost cell proliferates. These plasmids generally account for only asmall fraction of the total host cell DNA, and often carry genes thatconfer resistance to antibiotics. These genes, and the relatively smallsize of the plasmid DNA, are exploited in recombinant DNA technology.

The inserted DNA fragment of a recombinant DNA molecule may be derivedfrom an organism which does not exchange information in nature with thehost organism, and may be wholly or partially synthetically made.Construction of recombinant DNA molecules using restriction enzymes andligation methods to produce recombinant plasmids has been described inU.S. Pat. No. 4,237,224, issued to Cohen and Boyer. The recombinantplasmids thus produced are introduced and replicated in unicellularorganisms by means of transformation. Because of the generalapplicability of the techniques described therein, U.S. Pat. No.4,237,224 is hereby incorporated by reference into the presentspecification.

A different method for introducing recombinant DNA molecules intounicellular organisms is described by Collins and Hohn in U.S. Pat. No.4,304,863 which is also incorporated herein by reference. This methodutilizes a packaging/transduction system with bacteriophage vectors.

Because it is supercoiled, plasmid DNA can easily be separated from theDNA of the host cell and purified. For use as cloning vectors, suchpurified plasmid DNA molecules are cut with a restriction nuclease andthen annealed to the DNA fragment that is to be cloned. The hybridplasmid DNA molecules produced are then reintroduced into bacteria thathave been made transiently permeable to macromolecules (competent). Onlysome of the treated cells will take up a plasmid and these cells can beselected for the antibiotic resistance conferred on them by the plasmidsince they alone will grow in the presence of antibiotic. As thesebacteria divide, the plasmid also replicates to produce a large numberof copies of the original DNA fragment. At the end of the period ofproliferation, the hybrid plasmid DNA molecules are purified and thecopies of the original DNA fragments are excised by a second treatmentwith the same endonuclease.

Regardless of the method used for construction, the recombinant DNAmolecule must be compatible with the host cell, i.e., capable ofautonomous replication in the host cell. The recombinant DNA moleculeshould also have a marker function which allows the selection of hostcells transformed by the recombinant DNA molecule. In addition, if allof the proper replication, transcription and translation signals arecorrectly arranged on the plasmid, the foreign gene will be properlyexpressed in the transformed cells and their progeny.

2.2. VACCINES

Vaccines are an approach to the control and prevention of diseases.Vaccines can be prepared by mixing the immunogenic portion of an antigenwith an adjuvant. This preparation, when infected into a host animal orman, induces the production of antibody to the antigen, and thusprovides active immunization to the disease caused by the relevantorganism expressing the antigen.

Peptide vaccines contain only the necessary and relevant immunogenicmaterial, such as portions of the surface proteins of bacteria andviruses. Peptide vaccines can be made by isolating the relevant peptidefrom a highly purified bacterial fraction, or by synthesizing therelevant polypeptide. A major advantage of peptide vaccines is theexclusion of unrelated material of bacterial origin and of host- ordonor-derived interfering substances. However, at present, production ofpeptide vaccines using these methods is generally too expensive forwidespread commercial use. Recombinant DNA technology has much to offerin the production of peptide vaccines; the molecular cloning and hostcell expression of bacterial genes which encode the relevant immunogenicportions of the bacteria can produce sufficient quantities of therelevant immunogen for use in a peptide vaccine.

Vaccines are often administered in an emulsion with various adjuvants.The adjuvants aid in attaining a more durable and higher level ofimmunity using smaller amounts of antigen in fewer doses than if theimmunogen were administered alone. The mechanism of adjuvant action iscomplex and not completely understood. However, it may involve thestimulation of phagocytosis and other activities of thereticuloendothelial system as well as a delayed release and degradationof the antigen. Examples of adjuvants include Freund's adjuvant(complete or incomplete), Adjuvant 65 (containing peanut oil, mannidemonooleate and aluminum monostearate), and mineral gels such as aluminumhydroxide, aluminum phosphate, or alum. Freund's adjuvant is no longerused in vaccine formulations for humans or for food animals because itcontains nonmetabolizable mineral oil and is a potential carcinogen;however, the mineral gels are widely used in commercial veterinaryvaccines.

2.3. ATTEMPTS TO DEVELOP A STREPTOCOCCAL VACCINE USING M PROTEIN ANTIGEN

Fox, J. Immunol. 93:826-837 (1964) has used M proteins purified fromstreptococci as immunogens in rabbits to induce type-specificbactericidal antibodies. However, attempts at vaccinating humans withpartially purified streptococcal M proteins have been met with variedsuccess since strong local and systemic reactions usually occur inrecipients. See Schmidt, J. Infect. Dis. 106:250-255 (1960) and Potteret al., J. Clin Invest. 41:301-310 (1962). Fox et al., J. Infect. Dis.120:598-604 (1969) and Fox et al., J. Exp. Med. 124:1135-1151 (1966),using purified acid-extracted M protein, were partially successful withtheir vaccine. Of 22 adults vaccinated, 15 responded with a secondaryrise in type-specific antibody titer; however, only 5 exhibited a risein bactericidal antibodies.

Beachey et al., J. Exp. Med. 150:862-877 (1979) vaccinated 12 adultswith an alum precipitated preparation of a pepsin-derived fragment ofthe M24 protein (Pep M24). This was considered well tolerated since nolocal or systemic reactions were observed. Ten of the 12 personsvaccinated responded by developing M24 type-specific opsonic antibodies.

Immunological studies by Dale et al., J. Exp. Med. 151:1026-1037 (1980)revealed that 2 of the 12 volunteers, though immunized with M24, alsodeveloped antibodies that bind to both M5 and M6 proteins, of which onlythe M6 was opsonic. However, Beachy et al., in Symposium on BacterialVaccine, Ed. J. B. Robbins, J. C. Hill, Brian Decker Publisher, NewYork, pages 401-410 (1981), found that of four rabbits immunized withpurified Pep M5 protein (pepsin-derived fragment of the M5 protein), oneproduced antibodies directed against cardiac tissue in high titer. Thisantisera cross-immunoreacted with type M5 protein and cardiac tissues.

2.4. DNA HYBRIDIZATION ASSAYS

A general diagnostic method for the detection of pathogenicmicroorganisms may be devised if a DNA segment of the genome expected tobe found in such organisms is available in pure form. If it is, the DNAsegment may be used as a hybridization probe, by tagging it with achemical, enzymatic or radioisotopic reporter group.

Grunstein and Hogness Proc. Natl. Acad. Sci. U.S.A. 72:3961 (1975)! haveused this approach in a method called colony hybridization, wherebacteria to be assayed were transferred to a nitrocellulose filter. Thecolonies on the filter were then lysed, and the genomic DNA released wasfixed to the filter. The presence of nucleotide sequences in the affixedDNA that were complementary to the sequence of a ³² P-labeled probe wasthen monitored by autoradiography. Other general aspects of DNAhybridization have been described by Falkow et al. in U.S. Pat. No.4,358,535.

3. SUMMARY OF THE INVENTION

Methods and compositions are provided for the cloning and expression ofthe streptococcal M protein gene in single cell organisms. Alsodescribed are methods for culturing these novel single-cell organisms toproduce M protein, a rapid assay for identifying single colonies whichexpress the M protein DNA, and a method for identification of the geneproduct. The M protein produced by the recombinant DNA techniquesdescribed herein may be formulated for use as an immunogen in a vaccineto protect against Streptococcus pyogenes infection.

In a particular embodiment disclosed herein, the protein produced by E.coli transductants is slightly larger than the M6 protein isolated bysolubilization of the Group A streptococcal cell wall, but similar insize to that secreted by streptococcal protoplasts and L-forms.Immunologically, the molecule synthesized by E. coli transductants hasthe same type-specific determinants as the streptococcal M6 protein. TheM protein was characterized antigenically by Ouchterlony doublediffusion experiments and immunogenically by (a) an opsonic antibodyremoval test and (b) ability to elicit production of opsonic antibodies.The cloned M protein was isolated and then sized by sodium dodecylsulfate polyacrylamide gel electrophoresis. Additionally, methods aredescribed for isolating the expressed gene product.

The present invention provides a method of producing streptococcalopsonic antibodies and antigens of general importance in human medicineand in microbiological research. This includes use of streptococcal Mproteins produced by the present invention as highly reproduciblestandard antigens for ultrasensitive assays such as radioimmunoassays.These assays may be used as diagnostic tools for detection of antibodiesto streptococcus in biological samples.

Through the use of the streptococcal M protein gene or a fragmentthereof as a molecular probe, a method is also provided for thediagnostic identification of pathogenic streptococci in body tissues andfluids. In this method, DNA is extracted from microbial isolates andexamined for complementary nucleotide sequences by hybridization to themolecular probe. By this means large numbers of isolates may be screenedfor the presence of streptococci with relative ease, high sensitivityand accuracy. The result is a screening test offering marked advantagesover the more cumbersome serological diagnostic tests presently in use.

4. BRIEF DESCRIPTION OF THE FIGURES

The present invention may be more fully understood by reference to thefollowing detailed description of the invention and the figures inwhich:

FIG. 1 (not to scale) represents the construction of pJRS42, arecombinant plasmid derived from streptococcal DNA fragments and pJB8.(See Section 6.2. ) Various restriction sites within the streptococcalDNA are not shown.

FIG. 2 represents a restriction map of pJRS42.13. This plasmid wasderived from pJRS42 by digestion with EcoRI to remove unnecessarystreptococcal DNA, then religated. Plasmid pJRS42.13 has only a singleEcoRI site. (See Section 6.3. )

FIG. 3 is a restriction map of various subclones of plasmid pJRS42.13,which contains the genes coding for the M6 protein of type 6 Group Astreptococcus. The clear boxes define clones that express M6 protein,and the shaded boxes denote clones that do not. The vector pBR322 wasused in all cases except 42.21 and 42.19, which are in M13 and in pUC8and pUC9, respectively. The arrow above the restriction map indicatesthe direction of transcription of the gene encoding type 6M protein(emm6), and the approximate extent of the gene. The end of the moleculecorresponding to the amino terminus of the protein is denoted by "N".

FIG. 4 is a representation of a portion or the DNA sequence of the emm6gene that encodes the amino terminal end of the M6 protein, asdetermined by the method of Sanger et al. Proc. Natl. Acad. Sci. U.S.A.74: 5463 (1977)!, and the amino acid sequence of the protein aspredicted from the DNA sequence. The amino terminal amino acid, asdetermined by sequential Edman degradation (reference), is indicated byan "N" below the amino acid sequence.

FIG. 5 represents agar gel electrophoretic analysis of DNA hybridizationwith the NciI/PvuII emm6 DNA probe from the restriction enzyme digestionof plasmid pJRS42.13 of FIG. 3. The DNA in each lane is as follows:lane-l, oligonucleotide size standards of 10.90, 7.74, 5.15, 2.44, 1.80and 0.60 kb; lane 2, M6 strain D471; lane 3, M47; lane 4, M5; lane 5,M19; lane 6, M26; lane 7, M11; lane 8, M24; lane 9, M12; lane 10, M23;lane 11, M28 (from M-strain T28/51/4); and lane 12, M28 (from M+ strainT28/150A/5).

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. M PROTEIN IMMUNOGENS

This invention relates to the use of recombinant DNA techniques toproduce streptococcal M proteins which can be used as immunogens invaccine formulations. More specifically, the production of M6 protein isdescribed.

The recombinant plasmids, constructed as described herein, provide forhost cell (procaryotic or eucaryotic) production of a streptococcal hprotein which is stable and resistant to host cell degradation. Suchplasmids enable the generation of large quantities of protein, or afragment thereof, containing immunological and antigenic determinants ofnaturally-occurring M protein. The particular embodiment describedherein concerns M6 protein. However, the DNA molecules described hereinare not limited to the production of M6 protein and may be used toproduce any of the Group A streptococcal M proteins.

Generic applicability to all M proteins of the compositions and methodsdisclosed herein for M6 protein is apparent from the research ofFischetti and manjula (1982, Biologic and Immunologic Implications ofthe Structural Relationship Between Streptococcal M Protein andMammalian Tropomyosin, pp. 411-448 in Robbins, Hill and Sadoff (eds.),Bacterial Vaccines). For example, all of the M proteins sequenced sofar, including M5, M6 and M24, exhibit significant homology and all arecoiled-coil structures. The amino terminal and other sequenced segmentsof the three M molecules reveal that they all exhibit a repeatingseven-residue periodicity. Additionally, immunologic analysis of severalM types has revealed cross-reactions among various types. Fischetti, J.Exptl. Med. 146:1108-1123 (1977); Manjula and Fischetti, J. Exptl. Med.151:695-708 (1980); Manjula and Fischetti, J. Immunol. 124:261-267(1980). Furthermore, the hybridization data presented below indicatesstructural similarity among all 56M protein genes tested.

It can readily be seen by those skilled in the art that variousimmunogens and vaccine formulations can be prepared.

5.2. M PROTEIN MOLECULAR PROBE

The M protein gene described herein, or a fragment thereof, may be usedas d molecular probe in a diagnostic test for streptococci. The basis ofthe test is the fact that pathogenic streptococci possess gene sequencesthat are complementary to segments of the M protein gene of thisinvention. Such gene complementarity may be readily detected byisolating microbial DNA from a suspected streptococcal infection andannealing the DNA with the molecular probe under appropriate conditionson a solid support or in liquid medium. The occurrence of hybridizationmay readily be detected by attachment to the probe of a suitablereporter group.

Streptococcal organisms might be isolated from a point of infection inany body tissue or fluid, but a simple throat swab would be the mostlikely source. The microorganisms thereby obtained, which would probablybe a mixed culture, can be used directly (e.g., on the swab) or grownfirst to produce more cells in any of the culture media that are wellknown to those skilled in the art. Then, the DNA from the mixed culturecan be extracted, following disruption by freeze-thawing, sonication orother mechanical means and/or by treatment with general or specificagents that lyse bacterial cell walls.

The extracted DNA is generally denatured by the addition of aqueousalkali and then washed with a buffered solution. The particularconcentrations of alkali, buffer constituents, etc., depend on theconditions of the experiment and may be readily determined by routineexperimentation. The denatured M protein gene probe of this inventionmay then be added to the microbial DNA preparation and permitted tohybridize at points of DNA sequence complementarity. Specific bindingwill be recognized as that remaining after extensive washing of thehybridization mixture to eliminate non-specific binding.

The hybridization may be carried out in one of numerous solutions thathave been developed and are acceptable for the purpose. Falkow et al.have described many of the considerations of solution composition andthe hybridization process in general in U.S. Pat. No. 4,358,535, whichbecause of its general utility is hereby incorporated by reference. Thespecific values of other hybridization parameters, such as time andtemperature, and the method employed are not essential to thisinvention. Methods described by Gall and Pardue Proc. Natl. Acad. Sci.U.S.A. 63:376-383 (1969)! and by John et al. Nature 223:582-587 (1969)!,for example, can be applied. Indeed, it is expected that the method ofchoice for hybridization will change as the state of that art advances.

The molecular probe upon which the present invention is based mayencompass all or only a portion of the M protein gene, as long as enoughof the gene sequence is retained to permit specific hybridization tooccur. Such hybridization may be detected even at extremely low levelsby coupling an appropriate reporter group to the molecular probe. Theprobe may be radiolabeled (e.g. labeled with ³² P, ³ H, ¹⁴ C,³⁵ S etc.),or it may be tagged with a chemical or enzymatic reporter group. Forexample, colorimetric detectors coupled to biotinylated probes may beused in conjunction with avidin derivatives such as fluorescein-avidin,rhodamine-avidin, or enzyme-linked avidin.

The following Example of a method for the cloning and expression of theM6 protein in Escherichia coil and for the use of the M6 protein gene asa probe to characterize streptococcal infection is given for purposes ofillustration and not by way of limitation on the scope of the invention.

6. EXAMPLE PREPARATION OF THE CLONE CONTAINING THE STREPTOCOCCAL MPROTEIN GENE, AND USE OF THE GENE AS A MOLECULAR PROBE IN A DIAGNOSTICTEST FOR STREPTOCOCCAL INFECTION 6.1. ISOLATION OF STREPTOCOCCAL DNA

The source of the M6 protein gene was Streptococcus pyogenes strain D471(Group A streptococcus). Group C streptococcal phage lysin, in thepresence of 30% raffinose, was used to solubilize the Group Astreptococcal cell wall leaving stable protoplasts (Phillips et al.,Proc. Natl. Acad. Sci., U.S.A. 78:4689 (1981)). The protoplasts werethen washed extensively and treated with proteinase K to removestreptococcal DNAse. Protoplasts were lysed by dilution into sodiumdodecyl sulfate (SDS) and the extract was treated with ribonuclease I todigest RNA. Cesium chloride was added. The preparation was centrifugedat about 100×g to remove protein and dialyzed overnight. The DNA wasprecipitated with ethanol. The DNA fragments which were selected for usewere well over 100 kilobases (kb) before digestion (as assayed byagarose gel electrophoresis on a 0.4% agarose gel with P1 phage DNA as a100 kb standard and one-half P1 molecules as a 50 kb standard).

6.2. CLONING INTO E. COLI

A large piece of streptococcal DNA was to be cloned in order to reducethe number of candidate E. coli clones that needed to be screened for Mprotein production and to retain regulatory regions associated with thestructural gene for M protein. Therefore, a cosmid vector that acceptedDNA insertions of 35-40 kb was necessary. The 5.4 kb vector, pJB8, waschosen as the cloning vehicle. This vector had been constructed from theampicillin resistant plasmid HomerI and a synthetic BamHI linker in themanner described by Ish-Horowitz and Burke, Nucleic Acids Res.9(13):2989-2998 (191).

In the present invention, vector pJB8 was digested with BamHI asgenerally described by Maniatis et al., supra, pp. 104-106) whichcleaves the vector at a unique site to generate "cohesive ends". Thecleaved vector was treated with alkaline phosphatase (e.g., as inManiatis, et al., supra, 133-134) to remove the 5'-phosphates of thelinearized vector in order to prevent vector-vector religation andrecircularization. To generate random DNA fragments, Sau3a restrictionenzyme was used to partially digest the streptococcal DNA (e.g., as inManiatis, et al., supra, 298) isolated as described in Section 6.1. Thefragmented streptococcal DNA was ligated into the BamHI site on the pJB8vector (see FIG. 1, e.g., as in Maniatis, et al., supra, 298-299).

The vector with streptococcal DNA inserted was packaged into lambdaphage heads in vitro see Hohn and Collins, Gene 11:291 (1980)!. Thephage containing packaged chimeric DNA was used to transduce the E. coliK12 restriction-less strain C600NR(lambdacI857)recA which carries athermally inducible prophage. Ampicillin resistant colonies wereselected at 30° C., transferred to the same selective medium andincubated at 30° C. overnight to provide a master plate.

The possibility that the M protein gene would be expressed in E. coliwas high because most gram positive genes that have been cloned in E.coli have been expressed. However, it was unlikely that the M proteinwould appear on the surface of E. coli since it would have to betransported through the periplasm and the outer membrane, neither ofwhich exist in streptococcus. For that reason, the E. coli master platewas then shifted to 42° C. to induce prophage and lyse the host cells todetect expression of the cloned gene. Shalka and Shapiro, Gene 1:65(1976)!

6.3. RAPID ASSAY OF SINGLE COLONIES FOR EXPRESSION OF M PROTEIN

A rapid assay was developed to recognize the clone of E. coli expressingM protein. This technique readily distinguished single coloniesexpressing M protein.

The assay involved transferring the lysed colonies to be tested tonitrocellulose filters, rinsing the filters in bovine serum albumin toreduce non-specific affinity of the filter for proteins, reacting thefilter with antiserum to purified LysM6 that had been exhaustivelypre-absorbed with E. coli cells, rinsing appropriately, reacting with¹²⁵ I-Staphylococcal protein A (which binds to the antigen-antibodycomplexes), rinsing again, and scoring by autoradioyraphy.

To detect the M6 protein, the antiserum was diluted over 1000-fold.There was no detectable reaction with E. coli when the antiserums wasused at a 10-fold dilution (over 100 times more concentrated). By thismethod, production by an E. coli clone of less than 1% of the amount ofM6 protein produced in the parental streptococcus was detectable.

Among 335 colonies screened, one reacted strongly with antiserum topurified M6 protein. The chimeric plasmid present in this strain wasnamed pJRS42. The ability of this E. coli clone to produce the M6protein was stably maintained on subculture in ampicillin-containingmedium.

Plasmid pJRS42 was treated with EcoRI endonuclease which led to removalof a segment of streptococcal DNA to form pJRS42.13. This plasmidretained all necessary replication functions and the entire sequencecoding for the M6 protein with its promoter system.

6.4. IDENTIFICATION OF THE GENE PRODUCT

E. coli strain C600NR containing pJB8 and C600NR transductant containingpJRS42 were grown at 30° C. to late log phase in Todd-Hewitt broth (beefheart infusion broth) containing ampicillin. The cells were pelleted andwashed twice, and lysed with ethylene diaminetetraacetic acid(EDTA)-lysozyme followed by freezing in dry ice-ethanol and thawingquickly at 37° C. Following treatment with DNAse, the cellular debriswas removed by centrifugation at 10,000×g for 30 minutes and the extractwas passed through a 0.45 micron Millipore filter and dialyzed against50 mM ammonium bicarbonate.

The identification of M protein molecule produced in E. coli wasdetermined by immunoblot analysis. Equivalent protein concentrations, asdetermined by the Folin reaction Lowry et al., J. Biol. Chem. 193:265(1951)!, were applied to a 12% polyacrylamide gel containing SDS. Astandard preparation of purified M6 protein extracted from the type 6streptococcus by solubilization of the cell wall with phage lysin wasapplied to an adjacent well as control. After electrophoresis, theseparated proteins were transferred to nitrocellulose and unreactedsites on the filter were blocked using Tween 20 (polyoxyethylenesorbitan monolaurate), and the filter was incubated with antiserumdirected against M6 protein extracted by lysin from streptococcus thathad been absorbed with E. coli . An enzyme-linked immunoassay usingalkaline phosphatase conjugated to goat anti-rabbit IgG was used todetect the bound antibody. Bands were visualized using indoxyl phosphateas the alkaline phosphatase substrate and nitroblue tetrazolium as thechromophore by the method of Blake et al. anal. Biochem. 136:175-179(1984)!. The M6 antiserum reacted with both the M6 control and theextract of the E. coli clone containing pJRS42, but not with the extractfrom the parent E. coli strain containing only the pJB8 vector.

The molecular weight of the M protein produced by the E. coli clone wascompared by immunoblot analysis to a standard preparation of purifiedLysM6 protein. This molecule is the result of solubilization of the cellwall of the streptococcus with the enzyme phage lysin and purificationby column chromatography. It represents the largest M protein moleculeisolated from the streptococcal cell wall.

The M6 protein was purified as follows: The E. coli containing plasmidpJRS42.13 was treated with lysozyme in the presence of EDTA and 20%sucrose. This allowed the periplasmic contents to be released into thesurrounding fluid. Centrifugation of the organisms left the periplasmiccontents in the supernatant along with other E. coli associatedproteins. Using this technique it was found that the M protein was inhigh concentration in the periplasmic space and virtually absent fromthe cytoplasm. Thus, this method was used to generate the startingmaterial for the M protein purification.

The M protein in the crude periplasmic contents was purified from othercontaminating proteins by column chromatography. The crude periplasmicpreparation was dialyzed against 5 mM ammonium bicarbonate buffer pH 5.5and applied to a column of carboxymethyl cellulose. The column waswashed with three volumes of the same buffer and the adherent proteinseluted in one step with 100 mM sodium phosphate pH 7.0. The elutedprotein (containing the M protein) was applied directly to ahydroxylapatite column equilibrated in 25 mM sodium phosphate pH 7.0.Thecolumn was washed with 2 column volumes of 200 mM sodium phosphate pH7.0 and the adherent M protein was then eluted with 400 mM sodiumphosphate pH 7.0. This method resulted in a highly purified M proteinpreparation as determined by SDS-polyacrylamide gel electrophoresis andsequence analysis.

Amino terminal sequence analysis of the purified coli-synthesized M6protein yielded a single phenylthiohydantoin (PTH)-amino acid at eachdegradation step, verifying the homogeneity or the final preparation. Inaddition, the amino terminal sequence was found to be identical to thatof the LysM6 molecule through the first 35 residues sequenced, with theexception of the amino terminal residue. The E. coli molecule has anadditional arginine residue at the amino terminus which may be clippedoff during purification of the lysine molecule.

The purified LysM6 preparation exhibited a multiple banding patternpreviously observed with the M6 molecule, which is probably due todegradation during extraction and purification. The three major bandscorresponded to apparent molecular weights of 51,000, 52,000 and 53,000daltons. The size heterogeneity of the M6 preparation probably resultedfrom differences at the carboxy-terminal region of the protein since,during amino terminal sequence analysis of this preparation bysequential degradation, only a single amino acid residue was released ateach step. Since the bands from the pJRS42-containing clone that reactedwith anti-M6 antibodies are all larger (molecular weights 55,000,57,000, 59,000 daltons) than any from the streptococcal preparation,this suggested that pJRS42 contained the entire structural gene for theM6 protein. This was supported by the fact that this molecular sizecorrelated well with the reported size of M protein secreted fromprotoplasts and L forms of type 12 streptococci (molecular weight 58,000daltons). Thus, the proteins in the E. coli preparation may be closer tothe size of the intact native M molecule than those released bylysin-extraction of streptococcus.

In addition, the M protein isolated from secreting streptococcal L formsand protoplasts appeared more homogeneous. There are several possibleexplanations for the differences in mobilities of the E. coli protein(s)reactive with anti-M6 antibodies from those of the bands of the purifiedstandard M protein. For example: 1) The protein(s) may include a leadersequence that has not been removed in the E. coli but is normallyremoved in streptococci; 2) the LysM6 molecule may represent a cleavageproduct produced during attachment to the streptococcal cell wall; 3)the LysM6 molecule may be a partial degradation product produced duringpurification of the protein; 4) the E. coli protein(s) may be a "fusion"product produced from a promoter in the vector; 5) a weak translationalstart sequence that is unable to function in streptococcus may be activein E. coli; and/or 6) the "stop" condon normally functional instreptococcus may be suppressed by the supE mutation in the E. colistrain.

6.5. IMMUNOGENIC CHARACTERIZATION OF THE GENE PRODUCT

An Ochterlony immunodiffusion comparison of M protein of E. coli withthat extracted by lysin from streptococcus was performed. Well 1contained unabsorbed rabbit antiserum prepared against lysin-extractedM6 protein synthesized by the streptococcus; Well 2 contained purifiedlysin-extracted M6 protein; Well 3 contained M6 protein from the E. colistrain C600NR (pJRS42) which had been partially purified bychromatography on DEAE and CM cellulose. The reaction was performed in1% agar gel prepared at pH 8.6 in 50 mM barbitol buffer. The gel wasdried and stained with Coomasie blue.

The results of this double diffusion experiments (gel not shown)supported the conclusion that the extract of the E. coli carrying pJRS42(but not that of the plasmid without insert) contained a molecule withantigenic determinants common to at least some of those of streptococcalM6 protein. Thus, the M6 protein synthesized in E. coli has at leastsome of the same type-specific determinants as the M protein extractedfrom type 6 streptococci, although the E. coli product has a higherapparent molecular weight.

6.6. BACTERICIDAL EFFECT OF CLONED M PROTEIN

In order to determine if the M protein produced by the E. coli containedthe antigenic determinants necessary to remove opsonic antibodies fromboth rabbit and human opsonic antiserum, the following absorptionexperiment was performed.

Purified E. coli -synthesized M6 protein was lyophilized in two 30 μgaliquots. Rabbit type 6 opsonic antiserum (0.5 ml) was added to one, anda similar amount of human serum opsonic for type 6 streptococci wasadded to the other dried protein sample to form a solution. The tubeswere incubated at 37° C. for one hour and allowed to remain at 4° C.overnight. The precipitate that formed was centrifuged at 20,000×g andthe resulting supernatant was used in a bactericidal assay using type 6streptococci.

The indirect bactericidal assay was carried out as described originallyby Lancefield, J. Exptl. Med. 110:271 (1959). Heparinized whole humanblood from normal donors was used as a source of phagocytes. Dilutionsof type 6 streptococci (100 μl) were mixed with 400 μl of the humanblood in the presence or absence of either absorbed or unabsorbed serum(100 μl ). The mixture was rotated end over end at 37° C. for 3 hr. Thesurviving organisms were determined by pour plate method. Rotatedcontrols without antiserum were run to test the ability of thestreptococci to grow in the donor's blood.

The M protein produced by E. coli transductants removed the opsonicantibodies from both the rabbit and human sera. See Table 1. Thus, theanti-phagocytic determinants of the E. coli M protein function similarlyto those of the native M6 molecule.

                  TABLE 1                                                         ______________________________________                                        REMOVAL OF HUMAN AND RABBIT                                                   OPSONIC ANTIBODIES WITH                                                       E. COLI PRODUCED M6 PROTEIN                                                                 Colonies Found*                                                 Treatment       Rabbit anti-M6                                                                           Human anti-M6                                      ______________________________________                                        Inoculum        20         18                                                 No serum (control)                                                                            790        930                                                Unabsorbed serum                                                                              8          0                                                  Absorbed with E. coli M6                                                                      1800       2890                                               ______________________________________                                         *Numbers represent colony forming units as assayed by pour plate method       after the 3 hour rotation.                                               

6.7. PRODUCTION OF TYPE 6 OPSONIC ANTIBODIES

Production of type 6 opsonic antibodies in rabbits after immunizationwith purified E. coli -produced M6 protein was accomplished as follows.Antisera to the purified E. coli-produced M6 protein was prepared in NewZealand white rabbits. The primary inoculation consisted of 100 μg of M6protein emulsified with complete Freund's adjuvant and givensubcutaneously at multiple sites. The animals were boosted after 4 weekswith the same dose of the M6 protein in incomplete Freund's adjuvant.Animals were bled 10 days later.

As determined by bactericidal assay (described above), rabbits immunizedwith the E. coli M6 protein developed antibodies which allowedphagocytosis of type 6 streptococci. See Table 2.

                  TABLE 2                                                         ______________________________________                                        PRODUCTION OF OPSONIC ANTIBODIES IN RABBITS                                   IMMUNIZED WITH COLI M6 PROTEIN                                                              Colonies*                                                       ______________________________________                                        Inoculum        43                                                            No serum (control)                                                                            1112                                                          Immune serum    0                                                             ______________________________________                                         *Protocol is that described in Section 6.6., supra. Numbers represent         colony forming units as assayed by pour plate method after the 3 hour         rotation.                                                                

6.8. DIAGNOSTIC TEST FOR STREPTOCOCCI 6.8.1. PREPARATION ANDPURIFICATION OF AN M-GENE DNA PROBE

To locate the gene encoding type M6 protein (emm6), plasmid pJRS42.13,described in Section 6.3, supra, as subjected to digestion with variousrestriction enzymes or combinations of them. The DNA fragments thusobtained were sized by electrophoresis through 0.8% agarose gel asdescribed by Maniatis, et al., supra, 150-161, and then ligated intodifferent vectors. These recombinant vectors were transformed into an E.coli K12 bacterium that was lysogenic for a thermally inducible lambdaprophage and screened for the production of protein reactive withanti-M6 protein antiserum, as described in Section 6.4.

The results of M6 protein expression analysis for many of these clonedDNA fragments are shown in FIG. 3. The clones delineated by clear blocksproduced a protein that was reactive with anti-M6 antiserum, while thoseshown as shaded blocks did not. Although some of the reactive clonessuch as pJRS42.19 contained too little streptococcal DNA to encode theentire M6 protein, their expression products were evidently ofsufficient size to show antigenic reactivity with the polyclonalantiserum.

To learn more about the orientation of the emm6 gene within the clonedDNA fragments, the streptococcal DNA in plasmid pJRS42.19 was ligatedinto the BamHI sites of plasmids pUC9 and pUC8 Messing and Veira, Gene19:269-276 (1982)!. The relationship of these plasmids to each other issuch that inserted DNA is arranged in opposite orientations in theplasmids. Anti-M6 antiserum reacts with the products of both of theseclones, indicating that the M6 protein fragment is synthesized when thestreptococcal DNA is present in either orientation in these vectors. Itthus appears that the inserted streptococcal DNA carries its ownpromotor. It this conclusion is correct, pJRS42.19 should encode theN-terminus of the M6 protein.

Following ligation of the streptococcal DNA fragment of pJRS42.19 intoM13mp8 and mp9 Messing and Veira, supra!, the sequence of the insertedDNA was determined by the Sanger dideoxy method Sanger et al., Proc.Natl. Acad. Sci. U.S.A. 74:5463 (1977)!. The portion of the sequenceencoding the amino terminal end of the M6 protein, together with theamino acid sequence specified by it, is shown in FIG. 4. The aminoterminal amino acid, as determined by sequential Edman degradation(reference), is indicated by an "N" below the amino acid sequence. Theamino acid sequence determined in this way was identical to thatestablished for the amino terminus of M6 protein that had been extractedeither by pepsin treatment or by phage lysis from streptococcal strainD471 reference!. It was also identical to the amino terminal region ofthe M protein produced by recombinant DNA methodology in E. coli inSection 6.4, supra.

These results showed that the emm6 gene begins within the DNA fragmentcontained in pJRS42.19. Comparison of the DNA sequence with the aminoacid sequence shown in FIG. 4 further demonstrates that the N-terminusof M6 protein is at a point that is 32 bases to the left of the Nci Isite, as shown in FIG. 3.

The M6 protein produced in E. coli was shown by sodium dodecyl sulfatepolyacrylamide gel electrophoresis Fischetti et al., J. Exptl. Med.159:1083-1095 (1984)! to have an apparent molecular weight of 59,000daltons. This fact would place the other end of the gene sequence at ornear the PvuII site in FIG. 3. Further sequence analysis revealed thatthe nonsense codon terminating the protein, TAA, is located 38 bases tothe right of the PvuII site (Hollingshead et al., manuscript inpreparation).

A suitable probe containing a large portion of the emm6 gene wasprepared by treating plasmid pJRS42.13 with NciI and PvuII. The locationof this probe within the pJRS42.13 restriction map is indicated by theheavy arrow in FIG. 3. This probe fragment was purified byelectrophoresis in 0.8% agarose gel, electroeluted, and passed throughan Elutip-d column (Schleicher and Schuell) 6.8.2. ISOLATION OFBACTERIAL DNA

Phage lysin was used to lyse cells of Group A streptococci, by themethod of Fischetti et al. J. Exptl. Med. 133:1105 (1971)!. For otherstreptococci, overnight Todd-Hewitt-yeast broth cultures (beef heartinfusion broth with yeast extract) were diluted ten-fold and grown at37° C. to a cell concentration of about 5×10⁶ cells per ml. Glycine wasadded to a concentration of 3% (w/v), and the cells were incubated for 2additional hours at 37° C. The cells were then washed, sonicated twicefor 15-second pulses, and resuspended in 10 mM Tris-HCl buffer (pH 8.0)containing 30% (w/v) sucrose and 10 mg/ml lysozyme.

After incubation for 30 minutes at 37° C., ethylenediaminetetraaceticacid was added to a final concentration of 10 mM, and incubation wascontinued for an additional 30 minutes at 37° C. Protease K (0.1 mg/ml,source) and sodium dodecyl sulfate (1% w/v) were then added, thecomponents were mixed by gentle inversion, and incubation was continuedfor another 30 minutes at 37° C. Following this procedure the cellsuspensions, which displayed no turbidity, were extracted withphenol:chloroform (10:1) until no protein was visible at the interface.The extracted DNA was then precipitated with ethanol.

The bacterial strains examined include Staphylococcus aureus, B.subtilis (strain CU1065) Streptococcus pneumoniae and the followingstreptococcal strains from The Rockefeller University collection thatare standard typing and grouping strains, respectively, used to preparespecific antisera: M1, T1/195/2; M3, B930/61/5; M3R, D58X; M4,T4/95/RB5; M5, T5B/126/4;M6, S43/192/3; M8, C256/86/3; M11, T11/137/3;M12, T12/126/4 (COL 6); M14, T14/46/8; M15, T15/23/7; M18, J17C/55/4;M22, T22/146/1; M23, T23/102/RB5; M24, C98/135/2; M25, B346/136/1; M27,T27/87/1; M28, T28/150A/5; M29, D23; M30, D24/126/3; M31, J137/69/3;M32, C121/39/8; M33, C107/102/2; M36, C119/83/2; M37, C242; M38,C94/80/2; M39, C95/95/1; M40, C143/25/9; M41, C101/103/4; M42,C113/55/5; M43, C126/170/2; M46, C105/41/5; M47, C744/RB4/6/5; M48,B403/48/5; M49, B737/137/2; M50, B514/33/6; M51, A309/77/1; M52,A871/106/2; M53, A952/94/3; M54, A953/87/3; M55, A928/73/1; M56, A963;M57, A995/91/2; M58, D315/87/3; M60, D335/38/3; M63, D459/50/2; M66,D794/76/2; M67, D795/95/1; group A, J17A4; group B, 090R; group C, C74;group D, D76; group E, K131; group F, F68C; group G, D166B; group H,F90A; group L, D167A; group M, D168A"X"; group N, C559; group O, B361.The following group A M typing strains from the Center for DiseaseControl, Atlanta, Ga., were also used: M2, SS633; M9, SS754; M13, SS936;M17, SS631; M19, SS400; M34, SS134; M59, SS913 and M62, SS984.

6.8.3. DOT BLOT HYBRIDIZATION STUDIES

Dot hybridization was performed on the extracted DNA samples using theprocedure for detection of specific DNA sequences described by Kafatoset al. Nucleic Acid Res. 7; 1541-1552 (1979)!. For a probe, theNciI/PvuII DNA fragment containing most of the emm6 gene (Section 6.8.1)was used following labeling with ³² P by nick translation by the methodof Botchan et al. Cell 9: 269-287 (1976)!.

To carry out the hybridization, the DNA extracts from the variousmicrobial sources were denatured in 0.6N NaOH for 15 minutes at roomtemperature, and then for 10 minutes at 0° C. Then, the samples wereneutralized with 2M ammonium acetate, and aliquots of the DNas werespotted on Biodyne A 0.2 micron nylon filters (Pall Filtration Corp.,Glen Cove, N.Y.) in a Bethesda Research Laboratories manifold.Hybridization was carried out by adding at least 2×10⁶ cpm/filter of thenick-translated ³² P probe in buffer containing 1.8M Tris-HCl with 0.2MTris base, and the filters were maintained at 64° C. overnight. Thefilters were then washed 10 times with the same buffer at 64° C., dried,and subjected to autoradiography on Kodak XAR-5 film with anintensifying screen. The exposure was carried out at -80° C. for 2-4days.

A summary of the results of these studies is shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        DNA HYBRIDIZATION WITH THE emm6 PROBE                                         Bacterial  Strains Showing  Strains Showing                                   Type       Hybridization    No Hybridization                                  ______________________________________                                        Group A    1, 2, 3, 4, 5, 6, 8, 9,                                                       11, 12, 13, 14, 15, 17,                                                       18, 19, 22, 23, 24, 25,                                                       27, 28, 29, 30, 31, 32,                                                       33, 34, 36, 37, 38, 39,                                                       40, 41, 42, 43, 46, 47,                                                       48, 49, 50, 51, 52, 53,                                                       54, 55, 56, 57, 58, 59,                                                       60, 62, 63, 66, 67, 68,                                                       A486, A712, D366, D780                                             M.sup.-  Group A                                                                         J17A4, *A486 Var.                                                                              T28/51/4                                          Strains                                                                       Other      C, G             B, D, E, F, H, L,                                 Streptococcal               M, N, O                                           Groups                                                                        Other Gram None             Streptococcus                                     Positive                    pneumoniae                                        Organisms                                                                                                 Staphylococcus                                                                aureus                                                                        Bacillus                                                                      subtilis                                          ______________________________________                                         *Strain A486 Var. is a group A variant strain.                           

Overall, the dot blot test showed hybridization between the emm6 probeand DNA from 56/56 different M types of group A streptococcus, and from4 non-typable group A strains and from 2 strains previouslycharacterized as M⁻. No hybridization was seen with DNA from the grampositive organisms Staphylococcus aureus or Bacillus subtilis, fromstreptococcal Lancefield groups B, D, E, F, H, L, M, N or O, or fromStreptococcus pneumoniae. Hybridization was observed, however, withgroups C and G streptococcal DNA. This finding was not unexpected sincegroup C streptocci have occasionally been implicated in humaninfections, and some strains appear to have a molecule on their outersurfaces which is functionally similar to M protein Woolcock, Infect.Immun. 10:568 (1974)!.

Group G streptococci have also been reported to cause a wide range ofhuman infections. Although it is uncertain whether the virulence ofthese organisms is always due to the presence of an M-like cell surfaceprotein, it may be that it is. Examination of three strains of group Gstreptococci isolated from human infections revealed the presence in thestrains of a type 12M cell surface protein Maxted and Potter, J. Genl.Microbiol. 49:119 (1967)!.

Among the group A strains tested were three that are functionally M⁻(i.e., they do not produce protective M protein and are thusphagocytized). The DNA from two of these M⁻ strains neverthelesshybridized with the emm6 gene probe, showing that they retain at leastsome of the emm gene intact. Presumably, these strains are mutants whoseemm gene product is either nonfunctional or synthesized in reducedamount. The DNA from one M⁻ strain did not hybridize with the probe,suggesting that in that strain the emm gene was substantially deleted.

The results of the dot blot tests were confirmed by a study in which DNAfrom various M type group A streptococcal strains was extracted anddigested with NciI and HindIII. Samples of the resulting fragments ofDNA were then separated by agarose gel electrophoresis as described byManiatis et al., supra, 150-161, and hybridized with the emm6 ³²P-labeled probe, and the positions of the hybrid DNA fragments wererevealed by autoradiography. The results are shown in FIG. 5.

In FIG. 5, lane 1 represents 10.9, 7.74, 5.15, 2.44, 1.80 and 0.60 kbDNA molecular size markers that had been labeled with ³² p. Lanes 2through 10 contained the probe-hybridized NciI/HindIII fragments of DNAfrom streptococci of M types 6, 47, 5, 19, 26, 11, 24, 12 and 23,respectively. In each case, there were two or more DNA fragments thathybridized with the probe. As expected from the dot blot studies, DNAfrom M⁻ strain T28/51/4 (lane 11) did not hybridize, while that from M⁺strain T28/150A/5 (lane 12) did.

7. DEPOSITS OF MICROORGANISMS

The following listed E. coli strains carrying the listed plasmid havebeen deposited with the Agricultural Research Culture Collection (NRRL),Peoria, Ill., and have been assigned the following accession numbers:

    ______________________________________                                        E. coli strain                                                                              Plasmid     Accession Number                                    ______________________________________                                        K-12, C600NR  pJRS42.13   NRRL B-15529                                        Lambda cI857                                                                  K-12, C600NR  pJRS42      NRRL B-15535                                        Lambda cI857                                                                  ______________________________________                                    

The present invention is not to be limited in scope by themicroorganisms deposited, since the deposited microorganisms areintended to be illustrative of several aspects of the invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

In the present specification the base pair sizes given for nucleotidesare approximate and are used for purposes of description.

We claimed:
 1. An isolated polypeptide comprising a streptococcal M6protein.
 2. A polypeptide according to claim 1 wherein said polypeptideproduces opsonic antibodies to type 6 streptococci when injected intorabbits.
 3. A composition of matter comprising a polypeptide accordingto claim 1 and a carrier therefor.
 4. A polypeptide comprising astreptococcal M6 protein, said polypeptide having been produced by aunicellular organism by a process comprising the steps of introducing arecombinant plasmid having a DNA sequence coding for said polypeptideinto said organism, said plasmid being capable of being replicated,transcribed and translated by said organism, and culturing the organismin a nutrient broth.
 5. A polypeptide according to claim 4, wherein theunicellular organism is gram positive.
 6. A polypeptide according toclaim 4, wherein the unicellular organism is gram negative.
 7. Apolypeptide according to claim 4, wherein the unicellular organism isEscherichia coli.
 8. A polypeptide according to claim 4, wherein saidpolypeptide produces opsonic antibodies to type 6 streptococci wheninjected into rabbits.